The present invention relates to solid supports functionalized with phosphorus-containing dendrimers, to a process for preparing them, to their use for preparing biochips and to the uses of these biochips, in particular for immobilizing macromolecules, especially biological macromolecules such as nucleic acids, lipids, proteins (peptides, enzymes, antibodies, etc.) or molecular partners thereof.
The exponential development of genomics and pharmaco-genomics related to the sequencing not only of the human genome but also to that of animals, bacteria, viruses, plants, etc. is leading academic or industrial research laboratories to consume enormous amounts of reliable biochips that are easy to manufacture.
Now, in this particular context, it is of prime importance to have available functionalized solid supports that have a certain number of qualities.
These supports must in particular allow the reproducible immobilization of molecules of interest, since reproducible immobilization is conditional for detection that is itself reproducible.
These supports must also allow the immobilization of the molecules of interest in a sensitive manner. The sensitivity of a functionalized solid support depends on the degree of immobilization and on the method for detecting a signal, but also and above all on the level of background noise (nonspecific signal). A reduction in the background noise improves the signal/noise ratio. Specifically, in a device in which the presence of biological species in the region of the surface is detected, the background noise comes essentially from the nonspecific adsorption of molecules, including labeled biological molecules of interest, and should consequently be limited. The ideal is thus to obtain a support that has a very low background noise and a high signal detection intensity.
Moreover, and especially in the particular case of hybridization reactions using nucleic acids, the attachment of probes to the surface of a solid support should ensure the integrity of the sequence and the stability of the deposit. After the hybridization step, the results should be reproducible from one support to another. To this end, the probes should be spaced out on the surface of the solid support, so as not to disrupt the hybridization with the target nucleic acids. This makes it possible to come close to the solution hybridization conditions by mimicking a three-dimensional hybridization rather than a two-dimensional hybridization conventionally obtained via a glass-slide technique. A spacer of at least about 40 atoms (4-5 nm) is necessary to avoid the steric constraints between the DNA and the surface of the support.
Moreover, it is important to have available reusable functionalized solid supports. The possibility of having available a reusable support is of major interest since it permits the analysis of several biological samples with the same device, thus making it possible to make quantitative comparisons. Furthermore, the reusable supports enable several measurements to be taken on the same sample and thus allow a statistical improvement in the results.
At the present time, various types of processes for preparing biochips have already been proposed.
Biochips, and in particular nucleic acid biochips, may be manufactured either by in situ synthesis or by immobilization of probes.
In the first case (in situ synthesis), the probes are oligonucleotides, which are synthesized step by step directly on the support, and which are generally between 20 and 30 bases in size. This manufacturing process allows access to high-density chips (McGall et al, J. Am. Chem. Soc., 1997, 119, 5081-5090).
In the second case (immobilization of probes), the probes are presynthesized and then immobilized on the support. Two types of interactions with the surface may then take place.
The probe nucleic acid may be maintained on the surface by means of electrostatic interactions between the phosphate groups of the negatively charged skeleton and the modified surface of the positively charged support. By way of example, this type of biochip may be obtained by covering the surface with poly-L-lysine or by silanization with an aminosilane (Zammatteo et al, Anal. Biochem., 2000, 280, 143-150; Eisen et al, Methods in Enzymol, 1999, 303, 179-205). In this type of immobilization, the probe is generally coated onto the support, which may lead to problems of accessibility during the hybridization step. Furthermore, ionic interactions of this type are not sufficiently stable to allow reuse of the biochip.
The probe nucleic acid may also be grafted onto the support by means of covalent interactions. Generally, the bonds between the probes and the support are established at either the 3′ or 5′ end of the nucleic acid, allowing accessibility of the probe over its entire length, resulting in better quality of response in terms of hybridization. Several combinations of functionalization of the support and of the nucleic acids may be encountered. For example, the support may comprise nucleophilic functions such as —NH2 or —SH functions and the nucleic acid to be grafted may then comprise an electrophilic function, for instance a —CHO, —NCS, —NHS or —COOR function, and vice versa. According to another variant, the support and the nucleic acid may be nucleophilic and a di-electrophilic spacer will allow coupling between the two species. Finally, the support and the probe may be electrophilic and the coupling will be achieved by means of a di-nucleophilic spacer.
The biochips currently manufactured are, for the very large part, made using glass as support.
The biochips most frequently used have surfaces functionalized with a spacer ending with an —NH2 function. Their efficacy in terms of attachment and hybridization has been confirmed, but no reuse of this type of slide is possible, since the interactions between the probes and the support are of ionic type.
Other slides comprising aldehyde functions on their surface are marketed. These functions are introduced by using a silane-aldehyde, a simple spacer, which has only one anchoring function per molecule, whether it be on the support side or on the nucleic acid side. It has been shown that anchoring via several points to the surface is important (Zhao et al., Nucl. Acids Res., 2001, 29, 955-959; international patent application WO 01/51689) since it allows an increase in the sites of binding of the probes to the support, which results in a better response in terms of hybridization.
Another way of obtaining better detection sensitivity is reported especially in the studies by M. Beier and J. D. Hoheisel, Nucl. Acids Res., 1999, 27, 1970-1977. The authors constructed, on a glass slide, branched molecules containing six branches, with the aim of increasing the probe density. The nature of these branched spacers also makes it possible to modify the hydrophilic or hydrophobic nature of the surface. Their preparation process includes eight synthetic steps in total, starting from an amino slide, to obtain the biochip, which greatly limits their industrial development. Furthermore, the branched molecules are generated in situ in an uncontrollable manner, and their structure is not defined, which does not make it possible to ensure homogeneity of the surface of the biochip thus obtained. Since all the bonds are covalent, the authors have shown that their biochips could be reused. It should be noted that the reuse of biochips is common in the case of a Nylon® support and has more recently been described with plastic (see international patent application WO 00/55627).
Finally, recent studies (Benters et al, Chembiochem., 2001, 2, 686-694) describe the use of “PAMAM® starburst” amino dendrimers for the manufacture of biochips, seven synthetic steps that are demanding in terms of operating conditions, and of which some cannot be controlled since they are performed directly on the glass, for the production of a support that is not stable over time. Specifically, the use of these biochips includes a step of preactivation of the dendrimer layer before forming the coupling with the oligonucleotides. This step generates a reactive surface that is not stable over time, since the authors indicate that the probes must be grafted immediately after activation. This greatly limits the marketing of this type of support.
The exponential development of genomics and pharmacogenomics related to the sequencing not only of the human genome, but also to that of animals, bacteria, viruses, plants, etc. is leading academic or industrial research laboratories to consume enormous amounts of reliable biochips that are easy to manufacture. Reuse is an additional criterion that makes it possible to statistically validate biological tests. It is therefore in order to remedy all of these drawbacks and to have available reusable biochips that may be manufactured at low cost, in few steps, controllably and reproducibly, and that have excellent stability and very good detection sensitivity, that the Inventors have established the development that is the subject of the invention.